stereoselective and multiple carrier-mediated transport of cetirizine across caco-2 cell monolayers...

Stereoselective and Multiple Carrier-Mediated Transportof Cetirizine Across Caco-2 Cell Monolayers with

Potential Drug InteractionYING HE,1,2 YAO LIU,1 AND SU ZENG1*

1Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences,Zhejiang University, Hangzhou, Zhejiang, China

2Department of Biopharmaceutical Sciences College of Pharmacy, Univerisity of Illinois, Chicago, Illinois

ABSTRACT The aim of this study was to explore potential transport mechanisms ofcetirizine enantiomers across Caco-2 cells. Cetirizine displayed polarized transport atconcentrations ranging from 4.0 to 80.0 lM, with the permeability in the secretory direc-tion being 1.4- to 4.0-fold higher than that in the absorptive direction. Cetirizine enan-tiomers were transported distinctively different from each other. In the presence ofinhibitors of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP),the absorptive transport was enhanced and secretory efflux was diminished. When vera-pamil, indomethacin, or probenecid were present, the difference in the absorptive per-meability of R-cetirizine and S-cetirizine substantially intensified, whereas quinidinecould eliminate. R-cetirizine significantly increased the efflux ratio of rhodamine-123 anddoxorubicin in a fashion indicative of the upregulation of P-gp and MRP activities. How-ever, S-cetirizine played a role of an inhibitor for P-gp and MRP. Ranitidine modified theabsorption of cetirizine enantiomers, suggesting that the potential drug–drug interactionwould significantly change the cetirizine pharmacokinetics. In conclusion, the resultsindicated that there are several efflux transporters including P-gp and MRP participatingthe absorption and efflux of cetirizine, which showed enantioselectivity in the transmem-brane process. In addition, both P-gp and MRP functions could be modulated by cetiri-zine in chiral discriminative ways. Chirality 22:684–692, 2010. VVC 2009 Wiley-Liss, Inc.

KEY WORDS: P-gp; MRP; Caco-2 cells; cetirizine; enantioselective; drug interaction

INTRODUCTION

Cetirizine (Zyrtec1), the nonsedating antihistamine, iscommonly used as the first-line agent for the treatment ofseasonal and perennial allergic rhinitis and chronic idio-pathic urticaria. It is one of the second generation H1-his-tamine receptor antagonists that shows some significantadvantages beyond the first generation compounds.1 Ithas demonstrated excellent efficacy and a favorable safetyprofile related to its low metabolism and absence of car-diac effects.

Cetirizine is a racemic mixture of R-cetirizine (now avail-able under the trademark Xyzal1) and S-cetirizine. Inmost medicinal racemates, one of the enantiomers is moreactive than the other, and a number of studies have shownthat cetirizine and its enantiomers follow this rule.2 Opti-cally pure cetirizines show some stereoselective pharma-cological effects. S-cetirizine possesses potent activity intreating seasonal and perennial allergic rhinitis, the symp-toms of allergic asthma, chronic idiopathic urticaria, sometypes of physical urticaria, and other disorders includingthose that would benefit from an inhibitory action on eo-sinophil function, S-cetirizine, which is not presently com-mercially available is more useful for the treatment of urti-caria. It provides this effective treatment while avoiding

the concomitant liability of adverse effects associated withthe administration of racemic cetirizine by providing anamount which is insufficient to cause adverse effects asso-ciated with the administration of racemic. R-cetirizine ispreferred in the treatment of allergic disorders, and itavoids the adverse effects including, but not limited to,sedation and somnolence, headache, gastrointestinal dis-turbance, dizziness, nausea, cardiac arrhythmias, andother cardiovascular effects. These results are due to thehighly sensitive nature of antihistamine activity upon theprecise stereochemistry between the drug and histaminereceptor.3 R-cetirzine has demonstrated a twofold higheraffinity for the human H1-receptor compared with cetiri-zine, and �30-fold higher affinity than S-cetirizine did.4

Thus, the anti-H1 activity of cetirizine is primarily attributed

Contract grant sponsor: National Major Special Project for Science andTechnology Development of Ministry of Science and Technology ofChina; Contract grant number: 2009ZX09304-003*Correspondence to: Su Zeng, Department of Pharmaceutical Analysisand Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang Uni-versity, Hangzhou, Zhejiang 310058, China. E-mail: [email protected] for publication 19 November 2008; Accepted 21 October 2009DOI: 10.1002/chir.20815Published online 14 December 2009 in Wiley InterScience(www.interscience.wiley.com).

CHIRALITY 22:684–692 (2010)

VVC 2009 Wiley-Liss, Inc.

to R-cetirizine, the eutomer, whereas S-cetirizine can beconsidered as the distomer.

The therapeutic difference of cetirizine enantioners mayhave relation with the absorption, distrubution, metabo-lism, and excretion (ADME) profile of each enantiomer.Absorption within the gastrointestinal tract is the first stepgoverning the entry of drug in bloodstream and eventuallyits tissue distribution. Therefore, intestinal absorption andsecretion is of special importance and should be investi-gated to fully understand the mechanism which is respon-sible for the enantioselective pharmacokinetic propertiesof cetirizine. The pharmacokinetic properties of cetirizineenantiomers have been studied in vivo, but no work hasbeen reported on the comparative absorptive behaviors ofthe two enantiomers.5 In a previous in vitro studies, weinvestigated that R-cetirizine upregulates MDR1 expres-sion, whereas S-cetirizine downregulates MDR1 expres-sion.6 In this study, we investigated the intestinal absorp-tive differences between the two enantiomers in the Caco-2 cell model system.

Caco-2 cells originate from a human colorectal carci-noma and spontaneously differentiate on microporous fil-ter membranes into polarized monolayers. They acquiremany features of absorptive intestinal cells during culturesuch as microvillons structure, hydrolysis enzymes, andcarrier-mediated transport systems for sugar, amino acids,and several drugs.7 Caco-2 cells also express several effluxtransport proteins that may hamper drug’s absorption.These properties make the system particularly useful asan in vitro model for determining a drug’s absorptive char-acteristics, studying the transport mechanism of drugs,and elucidating their metabolism.8,9

Therefore, to evaluate the intestinal absorption andefflux mechanisms of cetirizine enantiomers, this studywas designed to study the transport characteristics of thetwo enantiomers using Caco-2 cell monolayers as a modelof human intestinal epithelium.

MATERIALS AND METHODMaterials

R-cetirzine and S-cetirizine were provided by HangzhouMinsheng Pharmaceutical Group Co., (Zhejiang, People’sRepublic China). Sodium dodecyl sulfate (SDS), Luciferyellow, cyclosporin A (CsA), sodium azide, verapamil, ci-metidine, quinidine, indomethacin, diclofenac, ranitidine,cefradine, doxorubicin, and rhodamine-123 were pur-chased from Sigma (St. Louis, MO). Probenecid waskindly provided by Shanghai Jicheng Pharmaceutical Plant(Shanghai, People’s Republic China). All solvents usedwere of HPLC grade and all chemical were of analyticalgrade.

Cell Culture

Caco-2 cells obtained from Chinese Academy of MedicalSciences (CAMS, Beijing, People’s Republic China) werecultured in high-glucose Dulbecco’s Modified Eagle’s Me-dium (DMEM, Gibco) supplemented with 10% fetal bovineserum (Gibco), 1% nonessential amino acid (Gibco), and

100 U/mL antibiotic–antimycolic solution. Cells weregrown in a humidified atmosphere of 5%CO2 at 378C.

After reaching 80% confluency, Caco-2 cells were har-vested with 0.25% trypsin-EDTA solution and seeded in12 mm i.d. Transwell1 inserts (catalog number 3460,Corning Coster Corp.) in 12-well plates at a density of 1.03 105 cells/cm2. Culture medium was replaced everyother day for the first 14 days and daily thereafter for next7 days until the monolayers expressed differentiated prop-erities that closely resemble morphologic and functionalcharacteristics of normal enterocytes.

Transport Experiments

Caco-2 cells in Transwells at passage 60–70 were usedfor transport experiments. The integrity of the monolayerwas checked by measuring transepithelial electrical resist-ance (TEER) value across the monolayer using Millicell-ERS voltohmmeter (Millipore) and by monitoring the per-meability of the paracellular leakage marker Lucifer yellowacross the monolayer.10 The cell monolayers were consid-ered tight enough for transport experiments when thePapp for Lucifer yellow was less than 0.2 3 1026 cm/s andTEER value >450 X cm2.

All transport studies were conducted at 378C unlessspecified otherwise. Before the experiment, the insertswere washed twice and preincubated for 30 min withwarm transport medium, Hank’s Balanced Salt Solutioncontaining 25 mM of HEPES, pH 7.4. Cetirizine enantiom-ers were dissolved in transport buffer into the desired finalconcentration ranging from 4.0 to 80.0 lM. The concentra-tion interval for cetirizine was chosen to cover the range ofluminal concentrations expected at the absorption site inthe human jejunum. The solutions were sterile filtered andadded on either the apical (AP, 0.5 ml) or the basolateral(BL, 1.5 ml) side of the inserts, whereas the receivingcompartment contained the corresponding volume oftransport medium. Transport studies were conducted inthe absorptive direction (AP?BL) and the secretory direc-tion (BL?AP), separately. After 1 h incubation, 0.5 mlsamples were collected from the receiving sides of the cellmonolayers for HPLC analysis.

Evaluation of Energy Depletion on the Transport ofCetirizine Enantiomers

To determine energy dependency of cetirizine transport,transport medium depleted in glucose was used in bothsides of the cell monolayers. ATP inhibitor, Sodium azide,was added to both AP and BL side and the monolayerswere incubated for 1 h at 378C.11 Transport experimentsalso performed at 48C to investigate the effect of tempera-ture on the transport of cetirizine enantiomers.

Evaluation of Carrier-Mediated Transport

To detect any significant carrier-mediated transport ofcetirizine and to evaluate the possible stereoselectivitycaused by transport protein, the bi-directional transportrates of R-cetirizine and S-cetirizine were determined inthe presence of several inhibitors. P-gp inhibitors (CsAand verapamil), the nonspecific inhibitors of MRPs (indo-methacin and probenecid), the organic cation transporter

685STEREOSELECTIVE TRANSPORT OF CETIRIZINE

Chirality DOI 10.1002/chir

(OCT) inhibitors (cimetidine), and the mixed inhibitor ofP-gp, MRP, and OCT (quinidine) were added to AP andBL compartments to investigate the inhibition of P-gp-,MRP-, and OCT-mediated transport of cetirizine enantiom-ers across Caco-2 cell momolayers.

The Influence of Cetirizine Enantiomers on theTransport of Typical P-gp and MRP Substrates

To investigate whether cetirizine enantiomes affect thefunction of P-gp and MRP, the transport characteristics ofP-gp substrate rhodamine-123 (5.0 lM) and MRP sub-strate doxorubcin (30.0 lM) were studied in the presenceof R-cetirizine or S-cetirizine (100.0 lM). Rhodamine-123was quantified using SPECTRA max M2 (Molecular De-vices) operating at excitation wavelength of 500 nm andemission wavelength of 540 nm. Similarly, doxorubcin wasquantified at excitation wavelength of 480 nm and emis-sion wavelength of 590 nm.

Investigate the Transport of Cetirizine Enantiomersin the Presence of Potential Clinicial

Coadministered Drugs

To explore the potential drug–drug interactions con-cerning cetirizine enantiomers, we examined the transportcharacteristics of R-cetirizine and S-cetirizine in the pres-ence of diclofenac potassium (0.37 mM), ranitidine (2.85mM), or cefradine (7.16 mM). Diclofenac potassium is apotent analgesic, nonsteroidal, anti-inflammatory drugs(NSAIDs). Ranitidine is a histamine H2-receptor antago-nist, which is widely used to treat and prevent ulcers inthe stomach and intestines. Cefradine, which belongs to agroup of cephalosporins, is an antibiotic medicine used totreat bacterial infections. It is highly possible that thesecommonly used drugs might be simultaneously adminis-tered with cetirizine. Therefore, we dissolved these drugsin the transport medium into the assumed drug concentra-tions in the gastrointestinal tract to determine the possibleinteractions concerning the absorption of cetirizine enan-tiomers.

Ion-Pairing RP-HPLC Analysis of Cetirizine Enantiomers

R-cetirizine and S-cetirizine in samples were quantifiedby ion-pairing RP-HPLC. To 0.5 ml of samples obtainedfrom transport study in Caco-2 cell monolayers, 20 ll ofdiazepam was added as the internal standard. SDS wasused as ion-pairing reagent. The HPLC conditions were asfollows: Shimadzu LC-2010C HPLC system with UV detec-tion at 230 nm, an Aglient Zorbax SB-C18 column (5 lm,150 3 4.6 mm I.D.) with an ODS guard column (10 lm,10 3 5 mm I.D.). The mobile phase consisted of a mixtureof acetonitrile/0.02 M phosphate buffer solution contain-ing 10 mM SDS and 1.5% TEA (pH 3.13) (54:46, v/v) at aflow rate of 0.75 ml/min. The injection volume was 100 ll.

Calculations and Statistics

Transport rate of each enantiomer was obtained accord-ing to eq. 1.

Permeability of each enantiomer was estimated by cal-culating Papp according to eq. 2.

The extent of the polarized transport was measured byefflux ration shown as eq. 3

V ¼ dQ

dt � A ð1Þ

Papp ¼ dQ

dt � A � C0ð2Þ

Efflux Ratio ¼ PappðBL-APÞPappðAP-BLÞ

ð3Þ

where C0 is the initial concentration in the donor compart-ment and A is the surface area of the monolayer. dQ/dt isthe rate of appearance of cetirizine enantiomers on thereceiving side Papp(BL-AP) and Papp(AP-BL) are the BL?APand AP?BL permeability of each enantiomer.

Results are given as mean 6 SD. The differences amongthe different groups were evaluated by a one- or two-wayanalysis of variance (ANOVA) with a post-hoc test (Dun-nett’s multiple comparion test). The differences in Papp ofenantiomers were evaluated using paired t-test. P < 0.05was considered to be statistically significant.

RESULTSValidation of HPLC Method

This work presented simple and reliable HPLC methodfor the determination of cetirizine enantiomers in thetransport medium. Representative chromatograms forcetirizine with internal standard in HBSS are shown in Fig-ure 1. Under the chromatographic conditions describedearlier, the total running time was within 15 min with ac-ceptable separation of the compounds of interest. Therewere no interferences from the matrix components andfrom the presence of drugs such as CsA, verapamil, cimeti-dine, quinidine, indomethacin, diclofenac, ranitidine, andcefradine.

Calibration curves were constructed by performing aregression linear analysis of the peak area ratios of theenantiomers to the internal standard versus the enan-tiomer concentrations. The calibration curves of eachenantiomer were linear over the concentration rangesfrom 0.10 to 2.00 lM, i.e., R-cetirizine, Y 5 2.500X 20.0995, r 5 1.000; S-cetirizine, Y 5 2.541X 2 0.0537, r 50.9995.

The intraday and interday precision and accuracy wereanalyzed at concentrations of 0.10, 0.50, 2.00 lM infive replicates within 1 day and on 5 consecutive days,respectively. Validation data indicated this method wassensitive and reliable with acceptable accuracy and preci-sion (Table 1). The limit of detection, defined as the lowestT1 concentration of cetirizine which can be detected(signal-to-noise ratio 3) for each enantiomer, was 0.01 lM.The limit of quantification, defined as the lowest concentra-tion of cetirizine which can be quantitatively determinedwith suitable precision and accuracy (signal-to-noise ratio

686 HE ET AL.


> 10) for each enantiomer, was 0.05 lM (RSD < 8.5%, n 55).

Polarized and Enantioselective Transport of Cetirizine

Transport of cetirizine enantiomers across Caco-2 cellmonolayers occurred in both AP?BL and BL?AP direc-tions. Cetirizine displayed polarized transport at concentra-tions ranging from 4.0 to 80.0 lM, with the secretory Papp

being 1.4- to 4.0-fold higher than the absorptive Papp. ThePapp (BL-AP) was independent on the concentration applied,whereas Papp (AP-BL) decreased with increasing concentra-tion, suggesting saturation of certain transporters. Theefflux ratio increased as the concentration increased.

As shown in Figure 2, the bi-directional transport of R-cetirizine and S-cetirizine were in different manner. In theabsorptive direction, the transepithelial transport rate of R-cetirizine was higher than S-cetirizine. Meanwhile, the se-cretory transport rate of cetirizine enantiomers did notshow any significant difference in the concentration range4.0 lM to 20.0 lM. With increasing concentration (40.0–80.0 lM), the distinctive transport difference betweencetirizine enantiomers was observed.

Several models to describe the kinetics of the ab-sorptive and secretory transport of cetirizine enantiomers(Michaelis-Menten model and Non-Michaelis-Mentenmodel, single and two binding sites, substrate inhibition,and the sigmoid models) were fitted and compared usingSigmaplot 9.0 (SPSS, Chicago, IL). The choice of modelwas confirmed by F-test and Akaike’s information crite-rion.12 It was found that in the absorptive transport, a

TABLE 1. Precision and accuracy date forcetirizine enantiomers

Concentrationadded (lmol/l)

Concentration found (lmol/l)

R-cetirizine RSD (%) S-cetirizine RSD (%)

Intra-day (n 5 5)0.100 0.100 6 0.004 4.5 0.098 6 0.005 4.90.500 0.500 6 0.009 1.8 0.497 6 0.011 2.32.000 2.001 6 0.010 0.5 1.989 6 0.016 0.8

Inter-day (n 5 5)0.100 0.099 6 0.006 6.5 0.094 6 0.007 7.70.500 0.501 6 0.015 3.0 0.494 6 0.016 3.22.000 2.004 6 0.030 1.5 1.982 6 0.028 1.4

Fig. 1. Representative chromatogram of blank transport medium (A)and the sample spiked with I.S. and cetirizine (B). 1: Diazepam (I.S.); 2:R- or S-cetirizine. [Color figure can be viewed in the online issue, which isavailable at www.interscience.wiley.com.]

Fig. 2. Transport rate of cetirizine enantiomers across Caco-2 cell mon-layers in the absorptive (A) and secretory (B) direction. The insets repre-sented the Eadie-Hofstee transformation of the data for the bi-directionaltransport. Each data point represents the mean 6 SD for three independ-ent monolayers.



dual-transporter Michaelis-Menten model (eq. 4) was thebest fit for both enantiomers. In the secretory direction, R-cetirizine and S-cetirizine followed the classic Michaelis-Menten model of one-binding site kinetics (eq. 5).

V ¼ Vmax1½S�Km1 þ ½S� þ

Vmax2½S�Km2 þ ½S� ð4Þ

V ¼ Vmax½S�Kmþ ½S� þ Kd � ½S� ð5Þ

Where V is the transport rate, [S] is the initial concentra-tion. Vmax is the maximum transport rate; Vmax1 and Vmax2

are the high- and low-affinity maximum transport rate,respectively. Km is the Michaelis-Menten constant; Km1

and Km2 are the high- and low-affinity Michaelis-Mentenconstants, respectively. Kd is the coefficient of nonsatura-ble transport.13

The transport kinetic parameters were shown in Table 1.In the absorptive direction, Km2 was more than 10 timeshigher than Km1 for each enantiomer. The in vitro clear-ance (CLint5 Vmax/Km, using only kinetic parameters forthe high-affinity component) demonstrated distinctive dif-ferences between enantiomers in the bi-directional trans-port. The clearance of S-cetirizine was higher than that ofR-cetirizine, which was consistent with the in vivo data.2

Effect of Temperature and ATP Inhibitoron the Transepithelial Transport

of Cetirizine Enantiomers

Bi-directional transport of cetirizine enantiomers acrossCaco-2 cell monolayers was investigated at both 378C and48C. The permeability of both enantiomers at 48C was sig-nificantly lower than at 378C. The polarized transport thatcan be obviously observed at 378C was almost disappearedwhen the temperature dropped to 48C. The lower tempera-ture also diminished the permeability difference betweenR-cetirizine and S-cetirizine. Transport experiment in thepresence of ATP inhibitor demonstrated that BL?APtransport rate of each enantiomer was decreased by almost36%. Furthermore, AP?BL transport of cetirizine did notshow any significant enantioselectivity (Fig. 3).

Effect of Potential Inhibitors on the TransepithelialTransport of Cetirizine Enantiomers

The polarized transport of cetirizine suggested that itwas likely to be mediated by some efflux transporters. Todetermine which carriers may be involved in the efflux ofcetirizine, a variety of inhibitors were used. The first groupwas CsA (10 lM) and verapamil (100 lM), typical inhibi-tors of P-gp. Inclusion of verapamil or CsA abolished theprofound polarized transport of cetirizine, with the Pap-p(AP-BL) for R-cetirizine and S-cetirizine increasing slightlyand the Papp(BL-AP) decreasing to a great extent. The sec-ond group was indomethacin (100 lM) and probenecid (2mM), the nonspecific inhibitors of MRPs. The resultsshowed that the transport rate in the secretory directionappeared to decrease by about 18%, whereas the absorp-

tive transport displayed distinct enhancement, which wasdifferent from the pattern of CsA and verapamil. The thirdgroup was cimetidine (50 lM), the inhibitor of OCT. Ci-metidine did not influence the transport of each enan-tiomer. Finally, quinidine (50 lM), the mixed inhibitor ofP-gp, MRP, and OCT,14 sharply raised the AP?BL trans-port rate demonstrating predominantly absorptive ratherthan secretory transport.

As demonstrated in Figure 4, verapamil, indomethacin,and probenecid substantially intensified the difference inthe absorptive permeability of R-cetirizine and S-cetirizine.Yet, the difference between R-cetirizine and S-cetirizinewas no longer manifested in the presence of quinidine.

Modulation of P-gp and MRPs Activityby Cetirizine Enantiomers

Since P-gp and MRPs might participate in the transportof cetirizine, the interplay of these important efflux pro-teins with cetirizine enantiomers was necessary to beexamined. Rhodamine-123 is selectively transported inCaco-2 cell monolayers by P-gp and is often used to studythe function of P-gp. As for dororubicin, it has beenreported as a MRP substrate.15 In this study, rhodamine-123 and doxorubicin were used as probes to study thecetirizine enantiomers on the P-gp and MRPs activity bydetermining the bi-directional transport of doxorubicin inthe presence of cetirizine enantiomers.

The results are shown in Figure 5. The control experi-ments demonstrated that this cell model system expressedadequate amounts of P-gp and MRP thus leading to a suffi-ciently adequent efflux ratio for rhodamine-123 and doxor-ubicin. In the presence of R-cetirizine, the efflux ratios ofrhodamine-123 and doxorubicin were enhanced by 48 and67%, respectively. On the other hand, the efflux ratios ofthese two substrates were reduced to 47 and 87% of theoriginal when S-cetirizne was presented. It was interestingto note that P-gp and MRP were vulnerable to inhibition oractivation by cetirizine in enantioselective way. R-cetirizine

Fig. 3. Effect of temperature and ATP inhibitor on the transport ofcetirizine enantiomers across Caco-2 cell monolayers. The experimentswere performed at concentration of 20.0 lM levocetirizine or dextrocetiri-zine. Each value represents the mean 6 SD for three independent mono-layers. ‘‘a’’ indicates Papp was significantly lower than the correspondingcontrol value, P < 0.05. ‘‘b’’ indicates Papp of levocetirizine was statisti-cally different from dextrocetirizine, P < 0.05.

688 HE ET AL.


played as an inducer/activator of P-gp and MRP, whereasS-cetirizine mannered as an inhibitor of these efflux trans-porters.

Potential Clinicial Coadministered Drugs Influence theTransport of Cetirizine Enantiomers

The interplay and modulation of efflux transporters withcetirizine enantiomers represent an important mechanismfor many clinically important drug–drug interactions.Diclofenac potassium, ranitidine, and cefradine were choseto investigate whether they would alter the absorption ofcetirizine enantiomers from the viewpoint of drug–drugand drug–protein interactions. The results in Figure 6show that diclofenac potassium and cefradine did not affectthe transport characteristics of cetirizine enantiomers. Thepolarized and enantioselective permeability of cetirizine didnot change significantly compared with the control experi-ment. In the presence of ranitidine, the absorptive transportrate of R-cetirizine and S-cetirizine boosted up by 15.7- and19.5-fold. Meanwhile, the seretory transport rate of R-cetiri-zine and S-cetirizine increased by 2.0-fold and 1.3-fold. Theabsorptive permeability was predominantly higher than thesecretory direction and the enantioselectivity was enlarged.The results indicated that the coadministration of cetirizinewith ranitidine could modify the absorption and the sys-temic toxicity of cetirizine.

DISCUSSION

This study demonstrated that cetirizine was predomi-nantly transported in the BL?AP direction in Caco-2 cellmonolayers. The polarized transport of cetirizine wasshown to be concentration-dependent and saturable. Thebiphasic and concave hyperbolic Eadie-Hofstee plots (Fig.2, insets) indicated that multiple carriers participated inthe transport process.11,16,17 The transport might occur inthe presence of high-affinity, low-capacity and low-affinity,high-capacity carrier systems.18 The model fitting resultsindicated that there might be two carriers participated inthe absorptive transport and one in the secretory trans-port.

Experiments performed at 48C or in the presence ofATP inhibitor indicate that the transport of cetirizine wasenergy-dependent. Temperature changes may affect thedrug diffusivity, membrane fluidity, transporter activity, in-tracellular trafficking, and other factors that may contrib-ute to the change in permeability. Furthermore, the lower

Fig. 4. Effect of inhibitors on transport of cetirizine enantiomers acrossCaco-2 cell monlayers Caco-2 cell monolayers were incubated at 378C for1 h with levocetirizine or dextrocetirizine added either to the apical side(A) or basolateral side (B) of the cell monolayers. CsA (10 lM), verapamil(100 lM), indomethacin (100 lM), probenecid (2 mM), cimetidine(50 lM), or quinidine (50 lM) was added to both the apical and basolat-eral side. Each value represents the mean 6 SD for three independentmonolayers. Significantly different between levocetirizine and dextrocetiri-zine by paired t-test, * P < 0.05, ** P < 0.01.

Fig. 5. Effect of levocetirizine and dextrocetirizine on the transport of rhodamine-123 (A) and doxorubicin (B) across Caco-2 cell monolayers. Rhoda-mine-123 (5.0 lM) and doxorubcin (30.0 lM) were added in the presence of levocetirizine or dextrocetirizine (100.0 lM). Each value represents themean 6 SD for three independent monolayers.



temperature will reduce the contribution of energy-de-pendent mechanisms such as P-gp efflux system to thedrug transport process.19 It is important to note thatreduced permeability with the presence of ATP inhibitorcan be attributed to inhibition of carrier-mediated transportmechanism. Therefore, these results are in agreementwith inhibition of ATP-dependent efflux pumps.

Since several efflux carriers work cooperatively in thetransport process, we try to elucidate the possible effluxtransporter(s) responsible for restricting the permeation ofcetirizine. Previous studies reported that cetirizine was thesubstrate of P-gp.20 Its affinity for P-gp at Blood-Brain Bar-rier (BBB) may explain the lack of central nervous systemside effects of this modern H1-antagonist. Therefore, theefflux transport of cetirizine in Caco-2 cell monolayers waspartly mediated by P-gp. Moreover, this study suggestedMRP besides P-gp might be implicated in transepithelialtransport of cetirizine. P-gp and MRP are two membraneproteins implicated in the active efflux of drugs and xeno-biotics. They belong to the ATP-binding cassette (ABC)superfamily of transport proteins and function as energy-dependent efflux pumps, decreasing free cellular concen-trations of drugs and xenobiotics. P-gp as well as MRP2 isexpressed at the apical side in Caco-2 cells, whereasMRP1 and MRP3 would be present at the basolateralside.21 The basolateral location of MPR1 and MPR3 makes

the interpretation of efflux transport data intricate sinceMRP1 or MRP3 is an absorptive efflux transporter,whereas P-gp and MRP2 are secretory efflux transporters.

In this study, we particularly investigated the stereose-lectivity in the transport of cetirizine across Caco-2 cellmonolayers. Living organisms are based on a plethora ofchiral molecules and often display different biologicalresponses to drug enantiomers. It is not uncommon forone enantiomer to be active, whereas the other is toxic inbiological systems. Thus, each enantiomer is necessary toundertake pharmacokinetic, metabolic, physiological, andtoxicological evaluation in the search for drugs withgreater therapeutic benefits and low toxicity. Biomem-brane permeability and cellular uptake of chiral drugs mayexist enantioselectivity if the intracellular micromoleculesinteract with the enantiomers in chiral discriminativeways. Caco-2 cell model system is very suited for thisstudy since the purpose of this study is to evaluate the ki-netic characteristics of transepithelial transport of eachenantiomer of cetirizine.

The results showed that R-cetirizine and S-cetirizinewere transported in different manners (Fig. 2, Table 2). Itwas likely that certain enantioselective transporter(s)might participate in the transport process. Therefore, weconsidered that MRPs and/or P-gp might be responsiblefor the enantioselective transport of cetirizine. In the pres-ence of verapamil, indomethacin, and probenecid, the dif-ference of the absorptive permeability between R-cetirizineand S-cetirizine substantially intensified in comparisonwith control experiments. One possible explanation for thephenomena is that cetirizine enantiomers might have dif-ferent binding sites on MRP with different affinities. Vera-pamil is a substrate for both P-gp and MRP-1 and indo-methacin and probenecid are the nonspecific MRP inhibi-tors. They might competitively inhibit the binding effect ofone enantiomer on the MRP(s), and thus caused the trans-port difference between R-cetirizine and S-cetirizine muchmore profound. A previous study reported there was nodifference in the BBB transport properties of cetirizine’stwo enantiomers in vivo, indicating that P-gp transport wasnot different between the two.22 However, we could notabsolutely exclude P-gp’s effort in the enantioselectivetransport of cetirizine because in the presence of quini-dine, the enantioselective transport was disappeared. Quin-idine combined the inhibition effect of P-gp, MRP, andOCT. We considered that cetirizine enantiomers could nolonger bind to these efflux transporters when the big mo-lecular structure of quinidine blocked the binding sites onthese proteins. Hence, the efflux transport of cetirizine

TABLE 2. Transport kinetic parameters of cetirizine enantiomers across Caco-2 cell monolayers

AP?BL R-cetirizine S-cetirizine BL?AP R-cetirizine S-cetirizine

Vmax1 (lM cms21) 35.0 22.1 Vmax (lM cms21) 10.1 2106.1Km1 (lM) 3.3 1.6 Km (lM) 3.9 166.8Vmax2 (lM cms21) 448.3 506.8 Kd (cms21) 10.8 0Km2 (lM) 97.6 179.4

Fig. 6. Effect of clinicial coadministered drugs on the transport of cetir-izine enantiomers. The experiments were performed at concentration of20.0 lM levocetirizine or dextrocetirizine in the presence of diclofenac po-tassium (0.37 mM), ranitidine (2.85 mM), or cefradine (7.16 mM). Eachvalue represents the mean 6 SD for three independent monolayers. ‘‘a’’indicates Papp was significantlyhigher than the corresponding controlvalue, P < 0.05. ‘‘b’’ indicates Papp of levocetirizine was statistically higherthan dextrocetirizine, P < 0.05.

690 HE ET AL.


was reversed and the difference between the two enan-tiomers was eliminated. To point out the exact mechanismfor the stereoselectivity was difficult because it is highlypossible that more than one carrier mediated the transportof cetirizine in chiral discriminative ways. Therefore,efforts are needed to establish cell lines transfected with ahuman gene that lead to a higher, purer expression of atransporter gene in future studies.

P-gp, first discovered in 1976 in tumor cells, is resistantto multiple anticancer drugs. It has been demonstratedthat the continuous exposure of tumor cells to some anti-cancer drugs induces P-gp in vitro and in vivo. It wasreported that aspirin enhanced P-gp expression in humanT lymphoma cells.23 Many studies also have demonstratedthat MRP is vulnerable to inhibition, activation, or induc-tion.24 This suggested that drugs other than anticancerdrugs might also modulate P-gp and MRP activity. In thisstudy, an interesting modulation effect by R-cetirizine andS-cetirizine was observed. R-cetirizine played as an in-ducer/activator of P-gp and MRP, whereas S-cetirizinemannered as an inhibitor of the efflux transporters. It ispossible that P-gp or MRP might interact with cetirizineenantiomers in entirely different manners. This interpreta-tion could also illuminate that MPR and/or P-gp mightaccount for the enantioselective transport of cetirizine. P-gp and MRP modulation may be useful in clinical therape-atics by improving the pharmacokinetic and pharmacody-namic properties of substrates for which efficacy would belimited by the efflux proteins.

The activity of P-gp and MRP significantly affect cetiri-zine pharmacokinetics from the viewpoints of drug–drugand drug–protein interactions. The enantioselectivityobserved in absorption might influence the pharmacologi-cal activity and pharmacokinetics of cetirizine. In thisstudy, we found ranitidine, a P-gp substrate, would modifythe transport characteristics of cetirizine enantiomers.Based on the broad substrate specificity and tissue distri-bution of P-gp, the distribution of P-gp substrates may sub-stantially be altered either intentionally or unintentionally.A number of clinically important drug–drug interactions,the mechanisms of which previously were unexplained orattributed solely to inhibition of cytochrome P450 (CYP),are mediated by P-gp or concomitantly through P-gp andCYP modulation.25 The coadministration of cetirizine withother P-gp substrates or inhibitors could modify theADME profile of cetirizine, especially the enantioselectivepharmacokinetics.

Cetirizine is a zwitterion at physiological pH and is wellabsorbed when given orally according to the biopharma-ceutical classification system (BCS). Previously, in vivoexperiments compared the plasma and urinary pharmaco-kinetic parameters of R- and S-cetirizine.5 The resultsshowed that R-cetirizine had a higher plasma AUC, ahigher Cmax, a longer terminal half-life (t1/2), a lower non-renal clearance, and a smaller volume of distribution (Vd).Our findings agreed with the enantioselective pharmacoki-netics of cetirizine, indicating that R-cetirizine was theeutomer owning better bioavailability than S-cetirizine.Furthermore, the different absorptive characteristicsbetween cetirizine enantiomers observed in this study

might account in part for the stereoselective ADME profileof this chiral drug.

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stereoselective and multiple carrier-mediated transport of cetirizine across caco-2 cell monolayers...

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